Redox and Proteomic Stress Responses in Biliary Disease

胆道疾病中的氧化还原和蛋白质组应激反应

基本信息

批准号：
10636916
负责人：
MICHAEL A PACK
金额：
$ 45.14万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-15 至 2026-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10636916
关键词：
Adult Affect Antioxidants Award Bile Duct Diseases Bile Duct Epithelium Bile fluid Biliary Biliary Atresia Biology Body Fluids CRISPR screen Caustics Cell Culture Techniques Cell Line Cell physiology Cells Characteristics Child Childhood Client Clinical Data Clustered Regularly Interspaced Short Palindromic Repeats Congenital atresia of extrahepatic bile duct Consumption Cyclic GMP DNA-Directed DNA Polymerase Data Correlations Detergents Development Disease Disease Outbreaks Duct (organ) structure Ductal Epithelial Cell Epidemic Epigenetic Process Epithelial Cells Exposure to Extrahepatic Genes Glutathione Glutathione Disulfide Glutathione Metabolism Pathway Goals Heat-Shock Response Heterogeneity Human In Vitro Incidence Injury Knowledge Laboratories Link Liver Failure Livestock Mass Spectrum Analysis Mediating Mediator Metabolism Mitochondria Modeling Molecular Molecular Chaperones Mus Mutate Mutation Organelles Outcome Oxidation-Reduction Oxidative Stress Pathogenesis Physiological Plants Play Population Predisposition Property Proteome Proteomics Publishing Reactive Oxygen Species Reduced Glutathione Regional Anatomy Research Risk Risk Factors Role Signal Transduction Stress System Toxic effect Toxin Variant Vertebrates Work Zebrafish analog bile duct bile salts biliary tract biological adaptation to stress cellular targeting cholangiocyte combat disorder risk experimental study genome-wide human disease in vivo intrahepatic liver transplantation macromolecule mouse model multicatalytic endopeptidase complex novel novel therapeutics oxidative damage prevent proteostasis proteotoxicity regional difference response

项目摘要

PROJECT SUMMARY Diseases of the biliary system comprise a significant percentage of the indications for liver transplantation in adult and pediatric populations. The pathogenesis of these disorders likely involves cellular responses to the harmful effects of bile, to which epithelial cells lining the bile ducts are continuously exposed. Bile toxicity arises largely from the oxidative damage caused by bile salts, whose detergent properties disrupt intracellular organelles, particularly mitochondria. Bile duct epithelial cells, also known as cholangiocytes, have evolved strategies for preventing bile toxicity in addition to having robust anti-oxidant defense systems, which are present in all cells to combat reactive oxygen species generated during normal metabolism. Work from my laboratory using the zebrafish system has identified regional susceptibilities of cholangiocytes to redox stress imparted by the plant toxin biliatresone, whose consumption is associated with epidemic biliary atresia (BA) in livestock. Our most recent studies indicate a comparable role for regional variantion in heat shock mediated proteostasis in cholangiocytes exposed to biliatresone, and that these responses can be modified by the activation of cGMP signaling. Importantly, there is compelling clinical data indicating the modulation of stress responses contributes to risk and outcomes in human BA, thus validating the biliatresone models we employ. The goal of this proposal is to study the mechanisms responsible for the variation in cholangiocyte stress responses using zebrafish, mouse and human cell culture models. In Aim 1, we will characterize the molecular determinants of the redox stress response in zebrafish intra-hepatic and extra-hepatic cholangiocytes, determine whether variation in these responses evolve from epigenetic factors that arise during biliary development, and correlate these findings with mammalian cholangiocyte biology using mouse models. In Aim 2, we will identify the cellular targets responsible for biliatresone-induced injury and stress in mammalian cholangiocytes using two approaches; first, via pull-down experiments using photo-activatable biliatresone analog; second, by conducting a genome-wide CRISPR positive selection screen for genes required for biliatresone toxicity. In Aim 3, we will explore links between cGMP signaling and proteostasis and how this affects susceptibility of cholangiocyte to injury using three approaches: first, by defining the downstream regulators of cGMP signaling that mediate its inhibition of biliatresone toxicity, second, by correlating these data with a comprehensive analysis of the cholangiocyte proteome, and how it is modified by biliatresone and cGMP signaling, third, defining the sub- cellular domains of cGMP signaling activity and its downstream mediators.

项目概要胆道系统疾病在肝移植适应症中占很大比例成人和儿童人群。这些疾病的发病机制可能涉及细胞对胆汁的有害影响，胆管内壁的上皮细胞持续暴露于胆汁中。出现胆汁毒性主要来自胆汁盐引起的氧化损伤，胆汁盐的去污剂特性会破坏细胞内的细胞器，特别是线粒体。胆管上皮细胞，也称为胆管细胞，已经进化除了拥有强大的抗氧化防御系统之外，还有预防胆汁毒性的策略在所有细胞中对抗正常代谢过程中产生的活性氧。我的实验室使用斑马鱼系统进行的工作已经确定了胆管细胞的区域敏感性植物毒素胆阿曲松造成的氧化还原应激，其消耗与流行性胆汁病有关家畜闭锁（BA）。我们最近的研究表明区域变异在热休克中具有类似的作用介导暴露于胆管细胞中的蛋白质稳态，并且这些反应可以通过以下方式改变： cGMP 信号的激活。重要的是，有令人信服的临床数据表明压力的调节反应有助于人类 BA 的风险和结果，从而验证了我们采用的比阿曲松模型。该提案的目标是研究胆管细胞应激变化的机制使用斑马鱼、小鼠和人类细胞培养模型进行反应。在目标 1 中，我们将表征分子斑马鱼肝内和肝外胆管细胞氧化还原应激反应的决定因素，确定这些反应的变异是否是由胆道发育过程中出现的表观遗传因素演变而来，以及使用小鼠模型将这些发现与哺乳动物胆管细胞生物学相关联。在目标 2 中，我们将确定使用两种方法确定哺乳动物胆管细胞中胆阿曲松诱导的损伤和应激的细胞靶标方法；首先，通过使用光激活双阿曲松类似物的下拉实验；第二，通过进行对胆阿曲松毒性所需基因进行全基因组 CRISPR 正选筛选。在目标 3 中，我们将探索 cGMP 信号传导与蛋白质稳态之间的联系，以及这如何影响胆管细胞对使用三种方法来抑制损伤：首先，通过定义介导其介导的 cGMP 信号的下游调节器其次，通过将这些数据与对胆碱酯酶的综合分析相关联来抑制双阿曲松毒性胆管细胞蛋白质组，以及它如何被胆阿曲松和 cGMP 信号传导修饰，第三，定义亚组 cGMP 信号传导活动的细胞结构域及其下游介质。